Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF-alpha and IFN-gamma by both the binder and the killer subsets but not by the free subset. IFN-alpha activated the secretion of IFN-gamma only, whereas IL-2 activated the secretion of both TNF-alpha and IFN-gamma by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF-alpha and IFN-gamma secretion in both the binder and the killer subsets, though IFN-gamma secretion was more pronounced in the binder subset. Activation of TNF-alpha and IFN-gamma secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-gamma. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF-alpha and IFN-gamma, whereas the free NK subset secretes little or no TNF-alpha and IFN-gamma following activation. These data suggest that the ability of NK cells to secrete TNF-alpha and IFN-gamma following activation correlates with the functional stage of maturation of NK cells.